Direct Regulation ofBacillus subtilis phoPRTranscription by Transition State Regulator ScoC

Abstract

Induction of the Pho response in Bacillus subtilis occurs when the P(i) concentrations in the growth medium fall below 0.1 mM, a condition which results in slowed cellular growth followed by entry into stationary phase. The phoPR promoter region contains three sigma(A)-responsive promoters; only promoter P(A4) is PhoP autoregulated. Expression of the phoPR operon is postexponential, suggesting the possibility of a repressor role for a transition-state-regulatory protein(s). Expression of a phoPR promoter-lacZ fusion in a scoC loss-of-function mutant strain grown in low-phosphate defined medium was significantly higher than expression in the wild-type strain during exponential growth or stationary phase. Derepression in the scoC strain from a phoP promoter fusion containing a mutation in the CcpA binding site (cre1) was further elevated approximately 1.4-fold, indicating that the repressor effects of ScoC and CcpA on phoP expression were cumulative. DNase I footprinting showed protection of putative binding sites by ScoC, which included the -10 and/or -35 elements of five (P(B1), P(E2), P(A3), P(A4), and P(A6)) of the six promoters within the phoPR promoter region. P(A6) was expressed in vivo from the phoP cre1 promoter fusion in both wild-type and scoC strains. Evidence for ScoC repression in vivo was shown by primer extension for P(A4) and P(A3) from the wild-type promoter and for P(A4) and P(E2) from the phoP cre1 promoter. The latter may reflect ScoC repression of sporulation that indirectly affects phoPR transcription. ScoC was shown to repress P(A6), P(A4), P(E2), and P(B1) in vitro.